Activation-induced deaminase (AID) belongs to the APOBEC family enzymes which convert cytosines in nucleic acids to uracil. AID is required for two processes essential for antibody maturation- somatic hypermutations and class-switch recombination Additionally, AID is also known to cause hypermutations in oncogenes and promote chromosome translocations that are hallmark of cancer. Among the unanswered questions regarding the role of AID in carcinogenesis is how AID selects certain genes and some loci for deamination. During the last funding period we studied the role played by transcription in the targeting process; in this proposal we will investigate how substrate selection by this enzyme affects the genetic outcomes. We will determine how the frequency, location and spectrum of genetic alterations changes when the substrate specificity of AID is altered. To accomplish this an AID- APOBEC3G hybrid with a strong preference for deaminating the last cytosine in a run of C's will be introduced in AID-/- murine B cells and the resulting hypermutations, isotype switching and chromosome translocations will be studied. The translocation of c-myc gene to one of the Ig genes is known to require AID and the junctions of such translocations promoted by mutant AID constructs will be identified and sequenced. AID will also be mutated to change its poor efficiency of converting 5-methylcytosine (mC) in DNA to thymine. Such conversions have been tied to DNA demethylation that is essential for reprogramming of embryonic cells and to translocation hotspots responsible for a number of human lymphoid malignancies. The demethylation activity of the mutants will be confirmed and AID mutants with altered preference for mC will be expressed in AID-/- B cells to determine whether the frequency, spectrum or locations of chromosome translocations are altered. These studies will create novel tools to study genetic instability promoted by AID and will answer specific questions regarding how the selection of DNA substrates by AID at nucleotide level influences its beneficial as well as harmful biological effects.